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Applied Microbiology and Biotechnology (v.58, #3)
Bacterial amino acid transport proteins: occurrence, functions, and significance for biotechnological applications by A. Burkovski; R. Krämer (pp. 265-274).
Transport processes play a pivotal role in cellular metabolism, e.g. for the uptake of nutrients or the excretion of metabolic waste products. Moreover, they are also important in biotechnological processes such as the production of various amino acids by the use of microorganisms. The focus of this review is on bacterial amino acid transport systems, in particular those of Corynebacterium glutamicum and Escherichia coli, with respect to their function and biotechnological significance.
Microbial production of vitamin B12 by J.-H. Martens; H. Barg; M. Warren; D. Jahn (pp. 275-285).
One of the most alluring and fascinating molecules in the world of science and medicine is vitamin B12 (cobalamin), which was originally discovered as the anti pernicious anemia factor and whose enigmatic complex structure is matched only by the beguiling chemistry that it mediates. The biosynthesis of this essential nutrient is intricate, involved and, remarkably, confined to certain members of the prokaryotic world, seemingly never have to have made the eukaryotic transition. In humans, the vitamin is required in trace amounts (approximately 1 µg/day) to assist the actions of only two enzymes, methionine synthase and (R)-methylmalonyl-CoA mutase; yet commercially more than 10 t of B12 are produced each year from a number of bacterial species. The rich scientific history of vitamin B12 research, its biological functions and the pathways employed by bacteria for its de novo synthesis are described. Current strategies for the improvement of vitamin B12 production using modern biotechnological techniques are outlined.
Importance of redox balance on the production of succinic acid by metabolically engineered Escherichia coli by S. Hong; S. Lee (pp. 286-290).
We had previously shown that succinic acid production in a pfl ldhA double mutant strain of Escherichia coli could be enhanced by amplifying the malic enzyme activity. However, recombinant E. coli NZN111 (F– Δpfl::Cam ldhA::Kan) harboring pTrcML, a plasmid containing the E. coli malic enzyme gene, produced a considerable amount of malic acid along with the desired product, succinic acid. To have an insight into the intracellular metabolism, metabolic control analysis was carried out. From the results of a simulation, it was predicted that supplying additional reducing power could enhance succinic acid production. More reduced carbon substrate sorbitol was thus examined for the possibility of matching the potential during succinic acid production. When NZN111 (pTrcML) was cultured in LB medium containing 20 g sorbitol/l under a CO2 atmosphere, 10 g succinic acid/l was produced. The apparent yield of succinic acid was 1.1 g succinic acid/g sorbitol, which is 85% of the maximum theoretical yield. Therefore, it was found that redox balancing was important for the enhanced production of succinic acid in metabolically engineered E. coli.
High-level ethanol production from starch by a flocculent Saccharomyces cerevisiae strain displaying cell-surface glucoamylase by A. Kondo; H. Shigechi; M. Abe; K. Uyama; T. Matsumoto; S. Takahashi; M. Ueda; A. Tanaka; M. Kishimoto; H. Fukuda (pp. 291-296).
A Strain of host yeast YF207, which is a tryptophan auxotroph and shows strong flocculation ability, was obtained from Saccharomyces diastaticus ATCC60712 and S. cerevisiae W303-1B by tetrad analysis. The plasmid pGA11, which is a multicopy plasmid for cell-surface expression of the Rhyzopus oryzae glucoamylase/α-agglutinin fusion protein, was then introduced into this flocculent yeast strain (YF207/pGA11). Yeast YF207/pGA11 grew rapidly under aerobic condition (dissolved oxygen 2.0 ppm), using soluble starch. The harvested cells were used for batch fermentation of soluble starch to ethanol under anaerobic condition and showed high ethanol production rates (0.71 g h–1 l–1) without a time lag, because glucoamylase was immobilized on the yeast cell surface. During repeated utilization of cells for fermentation, YF207/pGA11 maintained high ethanol production rates over 300 h. Moreover, in fed-batch fermentation with YF207/pGA11 for approximately 120 h, the ethanol concentration reached up to 50 g l–1. In conclusion, flocculent yeast cells displaying cell-surface glucoamylase are considered to be very effective for the direct fermentation of soluble starch to ethanol.
Purification of food-grade oligosaccharides using immobilised cells of Zymomonas mobilis by R. Crittenden; M. Playne (pp. 297-302).
Immobilised cells of the bacterium Zymomonas mobilis were used to remove glucose, fructose, and sucrose from food-grade oligosaccharide mixtures. Unpurified fructo-, malto-, isomalto-, gentio-, and inulin-oligosaccharides, containing total carbohydrate concentrations of 300 g l–1, were added to immobilised cells, in 100 ml batch reactors. No pH control or nutrient additions were required. Contaminating glucose, fructose, and sucrose within the mixtures was completely fermented within 12 h. The fermentation end products were ethanol and carbon dioxide. A minor amount of sorbitol was also produced as a fermentation by-product in the inulin–oligosaccharide mixture. No degradation of the oligosaccharides in the mixtures was observed.
Production of γ-linolenic acid by Cunninghamella echinulata cultivated on glucose and orange peel by H. Gema; A. Kavadia; D. Dimou; V. Tsagou; M. Komaitis; G. Aggelis (pp. 303-307).
A newly isolated strain of Cunninghamella echinulata grown on glucose produced significant quantities of biomass and cellular lipids in media with high C/N ratio. The oil yield from glucose consumed increased after nitrogen exhaustion in the growth medium, but γ-linolenic acid (GLA) content in cellular oil systematically decreased during the lipid accumulation process. When lipid accumulation was completed, GLA concentration in the cellular lipids progressively increased. The highest GLA production (720 mg/l) was achieved in medium with a C/N ratio equal to 163. C. echinulata was also able to grow on orange peel. The C/N ratio in the orange peel decreased from 50 to 26 during solid-state fermentation. Maximum oxygen uptake was observed during assimilation of reducing sugars, whereas a polygalacturonase activity was detected after reducing sugars had been exhausted. The maximum GLA production was 1.2–1.5 mg/g of fermented peel, calculated on a dry weight basis. After enrichment of the pulp with inorganic nitrogen and glucose, an increase in the production of oil and GLA was observed.
Single cell oil production by Yarrowia lipolytica growing on an industrial derivative of animal fat in batch cultures by S. Papanikolaou; I. Chevalot; M. Komaitis; I. Marc; G. Aggelis (pp. 308-312).
The growth of an oleaginous strain of Yarrowia lipolytica on an industrial fat composed of saturated free fatty acids (stearin) was studied. Lipid accumulation during primary anabolic growth was critically influenced by the medium pH and the incubation temperature. This process was independent of the nitrogen concentration in the culture medium, but was favored at a high carbon substrate level and at a low aeration rate. At pH 6 and a temperature of 28–33°C, 9–12 g/l of dry biomass was produced, whereas significant quantities of lipids were accumulated inside the yeast cells (0.44–0.54 g of lipid per gram of biomass). The strain showed the tendency to degrade its storage lipids, although significant amounts of substrate fat, rich in stearic acid, remained unconsumed in the culture medium. Y. lipolytica presented a strong fatty acid specificity. The fatty acids C12:0, C14:0, and C16:0 were rapidly incorporated and mainly used for growth needs, while C18:0 was incorporated with reduced rates and was mainly accumulated as storage material. Reserve lipids, principally composed of triacylglycerols (55% w/w of total lipids) and free fatty acids (35% w/w), were rich in stearic acid (80% w/w), while negligible amounts of unsaturated fatty acids were detected. When industrial glycerol was used as co-substrate, together with stearin, unsaturated fatty acid concentration in the reserve lipid increased.
β-Galactosidase from a cold-adapted bacterium: purification, characterization and application for lactose hydrolysis by S. Fernandes; B. Geueke; O. Delgado; J. Coleman; R. Hatti-Kaul (pp. 313-321).
The enzyme β-galactosidase was purified from a cold-adapted organism isolated from Antarctica. The organism was identified as a psychrotrophic Pseudoalteromonas sp. The enzyme was purified with high yields by a rapid purification scheme involving extraction in an aqueous two-phase system followed by hydrophobic interaction chromatography and ultrafiltration. The β-galactosidase was optimally active at pH 9 and at 26 °C when assayed with o-nitrophenyl-β-D-galactopyranoside as substrate for 2 min. The enzyme activity was highly sensitive to temperature above 30 °C and was undetectable at 40 °C. The cations Na+, K+, Mg2+ and Mn2+ activated the enzyme while Ca2+, Hg2+, Cu2+ and Zn2+ inhibited activity. The shelf life of the pure enzyme at 4 °C was significantly enhanced in the presence of 0.1% (w/v) polyethyleneimine. The pure β-galactosidase was also evaluated for lactose hydrolysis. More than 50% lactose hydrolysis was achieved in 8 h in buffer at an enzyme concentration of 1 U/ml, and was increased to 70% in the presence of 0.1% (w/v) polyethyleneimine. The extent of lactose hydrolysis was 40–50% in milk. The enzyme could be immobilized to Sepharose via different chemistries with 60–70% retention of activity. The immobilized enzyme was more stable and its ability to hydrolyze lactose was similar to that of the soluble enzyme.
Utilization of ATP-binding cassette exporter for hyperproduction of an exoprotein: construction of lipase-hyperproducing recombinant strains of Serratia marcescens by A. Idei; H. Matsumae; E. Kawai; R. Yoshioka; T. Shibatani; H. Akatsuka; K. Omori (pp. 322-329).
The Serratia marcescens extracellular lipase (LipA) is an enzyme applicable to enantioselective hydrolysis of racemic substrates. The enzyme is secreted through an ATP-binding cassette (ABC) exporter, the Lip system, encoded by the lipBCD genes. The S. marcescens recombinant carrying pLIPE121, which encodes the lipA gene in pUC19, exhibited a higher LipA production level than the wild-type strain. However, the level was lower than expected, and secretion was suggested to be a bottleneck. lipBCD plasmids were introduced into S. marcescens recombinants harboring lipA plasmids and the effectiveness of the lipBCD plasmids in elevating LipA productivity was investigated. S. marcescens strains harboring both lipA and lipBCD plasmids showed sevenfold greater extracellular LipA activity than the strain harboring the lipA plasmid alone. A high level of extracellular LipA production (1,300 kU/ml) and high plasmid stability (enough to carry out large-scale cultivation) were observed under non-selective conditions. Addition of L-proline and Tween 80 was effective in increasing cell growth of the recombinant, which led to high LipA production. In batch cultivation using a 30-l jar fermentor, LipA production was achieved at a high level of 5,200 kU/ml. This is the first report describing utilization of ABC exporter for the overproduction of an industrially important extracellular protein.
Role of the general stress response during strong overexpression of a heterologous gene in Escherichia coli by T. Schweder; H. Lin; B. Jürgen; A. Breitenstein; S. Riemschneider; V. Khalameyzer; A. Gupta; K. Büttner; P. Neubauer (pp. 330-337).
The strong overexpression of heterologous genes in Escherichia coli often leads to inhibition of cell growth, ribosome destruction, loss of culturability, and induction of stress responses, such as a heat shock-like response. Here we demonstrate that the general stress response, which is connected to the stress response regulator σS (σ38, rpoS gene product), is suppressed during strong overproduction of a heterologous α-glucosidase. The mRNA levels of the rpoS and osmY stress genes drastically decrease after induction of the strong overexpression system. It is shown that an rpoS mutation causes a significant loss of cell viability after induction of the expression system. Furthermore, it is demonstrated that an E. coli clpP mutant, which could be suggested to improve heterologous protein production, is not a good production host if a tac-promoter is used to control the expression of the recombinant gene. Data from this study suggest that the overexpression of the α-glucosidase was greatly decreased by sigma factor competition in the clpP mutant, due to the increased σS level in this mutant background.
The level of pyruvate-formate lyase controls the shift from homolactic to mixed-acid product formation in Lactococcus lactis by Rix C. Melchiorsen; Væver K. Jokumsen; J. Villadsen; H. Israelsen; J. Arnau (pp. 338-344).
Regulation of pyruvate-formate lyase (PFL) activity in vivo plays a central role in the shift from homolactic to mixed-acid product formation observed during the growth of Lactococcus lactis on glucose and galactose, respectively. Characterisation of L. lactis MG1363 in anaerobic batch cultures showed that the specific in vivo activity (flux) of PFL was 4-fold higher in L. lactis cells grown with galactose, compared with cells grown with glucose. The change in the PFL flux correlated with the observed variation in the PFL enzyme level, i.e. the PFL enzyme level was 3.4-fold higher in L. lactis cells grown on galactose than in those grown on glucose. To investigate whether a variation in the level of PFL was responsible for the shift in pyruvate metabolism, L. lactis strains with altered expression of pfl were constructed. The pfl gene was expressed under the control of different constitutive promoters in L. lactis MG1363 and in the PFL-deficient strain CRM40. Strains with five different PFL levels were obtained. Variation in the PFL level markedly affected the resulting end-product formation in these strains. During growth on galactose, the flux towards mixed-acid products was to a great extent controlled by the PFL level. This demonstrates that a regulated PFL level plays a predominant role in the regulation of the metabolic shift from homolactic to mixed-acid product formation in L. lactis.
Implication of manganese (III), oxalate, and oxygen in the degradation of nitroaromatic compounds by manganese peroxidase (MnP) by B. Van Aken; S. Agathos (pp. 345-351).
The fungal ligninolytic enzyme manganese peroxidase (MnP) is known to function by oxidizing Mn(II) to Mn(III), a powerful oxidant. In this work, an abiotic system consisting of Mn(III) in oxalate buffer under aerobic conditions (Mn(III)/oxalate/O2 system) was shown to be capable of extensively transforming 2-amino-4,6-dinitrotoluene (2A46DNT) – one of the main reduction products of 2,4,6-trinitrotoluene (TNT). No significant transformation occurred in the presence of other organic acids or under anaerobic conditions. The Mn(III)/oxalate/O2 system was also able to transform other nitroaromatic compounds such as 2-nitrotoluene, 4-nitrotoluene, 2,4-dinitrotoluene, TNT – the latter to a lesser extent –, and their reduction derivatives. The Mn(III)/oxalate/O2 system mineralized 14C-U-ring labeled 2A46DNT slightly, while no significant mineralization of 14C-U-ring labeled TNT was observed. Unidentified 14C-transformation products were highly polar. Electron spin resonance experiments performed on the Mn(III)/oxalate/O2 system revealed the generation of formyl free radicals (·COO–). The oxygen requirement for the transformation of nitroaromatic compounds suggests the involvement of superoxide free radicals ( % MathType!MTEF!2!1!+- % feaaeaart1ev0aaatCvAUfKttLearuavTnhis1MBaeXatLxBI9gBam % XvP5wqSXMqHnxAJn0BKvguHDwzZbqegm0B1jxALjhiov2DaeHbuLwB % Lnhiov2DGi1BTfMBaebbnrfifHhDYfgasaacH8YjY-vipgYlH8Gipe % c8Eeeu0xXdbba9frFj0-OqFfea0dXdd9vqaq-JfrVkFHe9pgea0dXd % ar-Jb9hs0dXdbPYxe9vr0-vr0-vqpWqaaeaabiGaaiaacaqabeaada % abauaaaOqaaiabb+eapnaaDaaaleaacqaIYaGmaeaacqGHsislkiab % gwSixdaaaaa!3F2C! $$ {
m O}_2^{ - cdot } $$ ), produced through autoxidation of ·COO– by molecular oxygen. The implication of such a Mn(III)/oxalate/O2 system in the MnP-catalyzed degradation of nitroaromatic pollutants by white-rot fungi is further discussed.
Modeling growth and biochemical activities of Azospirillum spp. by I. Kefalogianni; G. Aggelis (pp. 352-357).
An unstructured mathematical model was developed and used in the evaluation of biochemical activities of four Azospirillum spp. strains grown in batch cultures in a high C/N-ratio medium. The strains were evaluated for their ability to grow on fructose and produce exo-polysaccharide, and to sustain nitrogenase activity by using fructose or polysaccharides. Quantitative expression of the regulation of polysaccharide synthesis and nitrogenase (acetylene reduction) activity from the mineral nitrogen and sugar concentration in the culture medium was achieved. It was found that, during growth, Azospirillum spp. produced significant quantities of exo-cellular and capsular polysaccharide, whereas after depletion of the carbon source from the culture medium polysaccharides were consumed, especially in A. lipoferum strains. Significant nitrogenase activity was detected during polysaccharide degradation. Oxygen uptake was high during assimilation of fructose and low during polysaccharide degradation.
Yield production, chemical composition, and functional properties of emulsifier H28 synthesized by Halomonas eurihalina strain H-28 in media containing various hydrocarbons by F. Martínez-Checa; F. Toledo; R. Vilchez; E. Quesada; C. Calvo (pp. 358-363).
Halomonas eurihalina strain H-28 is a moderately halophilic bacterium that produces an extracellular polysaccharide not only in media with glucose but also in media supplemented with hydrocarbons (n-tetradecane, n-hexadecane, n-octane, xylene, mineral light oil, mineral heavy oil, petrol, or crude oil). In this study we investigated yield production, chemical composition, viscosity, and emulsifying activity of exopolysaccharides (EPS) extracted from the different media used. The largest amounts of biopolymer were synthesized in media with glucose and n-hexadecane. Chemical composition varied with culture conditions; thus EPS from cultures grown in the presence of hydrocarbons had lower contents of carbohydrates and proteins than EPS from media with glucose. However, the percentages of uronic acids, acetyls, and sulfates were always higher than glucose EPS. Crude oil was the substrate most effectively emulsified. All EPS were capable of emulsifying crude oil more efficiently than the three control surfactants tested (Tween 20, Tween 80, and Triton X-100). All polymers gave low viscosity solutions. EPS H28 could be attractive for application in the oil industry and/or in bioremediation processes, bearing in mind not only its functional properties, but also the capacity of producer strain H-28 to grow in the presence of high salt concentrations and oil substrates.
Degradation of biphenyl by Mycobacterium sp. strain PYR-1 by J. Moody; D. Doerge; J. Freeman; C. Cerniglia (pp. 364-369).
The metabolism of biphenyl by Mycobacterium sp. PYR-1 was investigated. The Mycobacterium sp. degraded >98% of the biphenyl added within 72 h. Analysis of ethyl acetate extracts of the culture medium by HPLC indicated that benzoic acid was the major metabolite. Other products were 4-hydroxybiphenyl, 4-hydroxybenzoic acid, and 5-oxo-5-phenylpentanoic acid. The metabolites were characterized by mass and 1H NMR spectrometry. Identification of benzoic acid and 5-oxo-5-phenylpentanoic acid indicates that biphenyl degradation by Mycobacterium sp. PYR-1 is generally similar to known pathways. A novel alternative metabolic pathway consisted of monooxygenation at C-4 of biphenyl to give 4-hydroxybiphenyl, with subsequent degradation via ring cleavage to 4-hydroxybenzoic acid.
Analysis of alkali-soluble glucan produced by Saccharomyces cerevisiae wild-type and mutants by C. Ha; K. Lim; Y. Kim; S. Lim; C. Kim; H. Chang (pp. 370-377).
The alkali-soluble glucan of the yeast cell wall contains β-(1,3)- and (1,6)-D-linkages and systemically enhances the immune system. To isolate Saccharomyces cerevisiae mutants producing glucan with a high degree of β-(1,6)-D-glycosidic bonds, a wild-type strain was mutagenized with ultraviolet light. The mutants were then selected by treatment with 1.0 mg laminarinase, endo-β-(1,3)-D-glucanase/ml. The alkali-soluble glucan was extracted by modified alkali ysis followed by the Cetavlon method and concanavalin-A chromatography. The prepared alkali-soluble glucans from the wild-type and the mutants were compared with respect to yield and polymer structure using gas chromatography, 13C-NMR spectrometry, high performance liquid , and multi-angle laser light scattering and refractive index detectors. The results indicated that the S. cerevisiae mutants had ten-fold more alkali-soluble glucan than the wild-type. Structural analysis revealed that the alkali-soluble glucan from the mutants also had a higher degree of β-(1,6)-D-linkage than that from the wild-type.
Responses of Mycobacterium sp. LB501T to the low bioavailability of solid anthracene by L. Wick; A. de Munain; D. Springael; H. Harms (pp. 378-385).
Several recent reports have indicated that some bacteria may have adapted to the low bioavailability of hydrophobic environmental chemicals and that generalizations about the bioavailability of compounds such as polycyclic aromatic hydrocarbons (PAHs) may be inappropriate. Experimental evidence and theoretical considerations show that the utilization of PAHs requires bioavailability-enhancing mechanisms of the bacteria such as: (1) high-affinity uptake systems, (2) adhesion to the solid substrate, and (3) biosurfactant excretion. We examined possible specific physiological responses of anthracene-degrading Mycobacterium sp. LB501T to poorly water-soluble anthracene in batch cultures, using solid anthracene as a sole carbon source. Mycobacterium sp. LB501T exhibited a high specific affinity for anthracene (a o A=32,500 l g–1 protein h–1) and grew as a confluent biofilm on solid anthracene present as sole carbon source. No biofilm formation on anthracene was observed when excess glucose was provided as an additional substrate. This difference could be attributed to a modification of the cell surface of the bacterium. Anthracene-grown cells were significantly more hydrophobic and more negatively charged than glucose-grown cells. In adhesion experiments, anthracene-grown cells adhered 1.5- to 8.0-fold better to hydrophobic Teflon and up to 70-fold better to anthracene surfaces than glucose-grown cells. However, no production of biosurfactants was observed. Our results thus indicate that attachment and biofilm formation may be a specific response of Mycobacterium sp. LB501T to optimize substrate bioavailability.
Growth rate-dependent changes in Escherichia coli membrane structure and protein leakage by A. Shokri; A. Sandén; G. Larsson (pp. 386-392).
The phospholipid and fatty acid content of the Escherichia coli membrane were investigated during continuous cultivation. At low growth rates, there was an increase in cardiolipin produced at the expense of phosphatidylethanolamine. Phosphatidylglycerol had a maximum at a growth rate of 0.3 h–1. The amount of cyclic fatty acids was markedly increased at lower growth rates, while there was an evident minimum at 0.3 h–1. This was also the case for saturated fatty acids. At this point, the unsaturated fatty acids had a maximum depending mainly on changes in cis-vaccenic acid. The mechanical strength towards sonication and osmotic shock/enzymatic treatment showed that the cells were more rigid at low dilution rates. However, this was accompanied by a higher cell lysis, a reduced capacity for total and specific protein production and a lower yield of cells. The amount of lipid A in the medium (endotoxin) was constant and negligible at all growth rates. The leakage of periplasmic protein to the medium had an optimum at 0.3 h–1, resulting in a transport of 20% of the total recombinant product. It is argued that this constitutes the point of highest membrane fluidity and thus an increase possibility for protein transport.
Effects of randomly methylated-β-cyclodextrins (RAMEB) on the bioavailability and aerobic biodegradation of polychlorinated biphenyls in three pristine soils spiked with a transformer oil by F. Fava; V. Ciccotosto (pp. 393-399).
The low bioavailability of polychlorinated biphenyls (PCBs) in soils often results in their slow and partial aerobic biodegradation. The process can be enhanced by supplementing soils with cyclodextrins. However, pure cyclodextrins are expensive and we have therefore explored the use of a less costly technical-grade mixture of randomly methylated-β-cyclodextrins (RAMEB). RAMEB was tested at 0, 1, 3 and 5% (w/w) in the aerobic bioremediation and detoxification of a loamy-, a humic- and a sandy-soil, each artificially contaminated with a PCB-containing transformer oil (added PCBs: about 450 or 700 mg/kg), inoculated with an exogenous aerobic PCB-biodegrading bacterial co-culture and treated in slurry- and solid-phase laboratory conditions. Significant depletions of the spiked PCBs were observed in all microcosms of the three soils after 90 days of treatment; however, interesting yields of PCB dechlorination and detectable decreases of the original soil ecotoxicity were observed in the slurry-phase microcosms. RAMEB generally enhanced PCB-metabolism with effects which were dependent on the concentration at which it was applied, the physical-chemical nature of the amended soil, and the soil treatment conditions employed. RAMEB, which was slowly metabolized by soil microorganisms, enhanced the presence of PCBs and PCB-cometabolizing bacteria in the soil–water phase, suggesting that RAMEB enhances aerobic biodegradation of PCBs by increasing pollutant bioavailability in soil microcosms.
Inhibition of microbial H2S production in an oil reservoir model column by nitrate injection by S. Myhr; B.-L. Lillebø; E. Sunde; J. Beeder; T. Torsvik (pp. 400-408).
The effect of nitrate addition on microbial H2S production in a seawater-flooded oil reservoir model column with crude oil as carbon and energy source was investigated. Injection of 0.5 mM nitrate for 2.5–3.5 months led to complete elimination of H2S (initially 0.45–0.67 mM). The major decline in H2S level coincided with the first complete nitrate consumption and production of nitrite. When nitrate was excluded, H2S production resumed after approximately 2.5 months and reached previous levels after approximately 5 months. Using a fluorescent antibody technique, three populations each of sulfate-reducing bacteria (SRB) and nitrate-reducing bacteria (NRB) were monitored. SRB dominated the anoxic zone prior to nitrate addition, comprising 64–93% of the total bacterial population. The monitored NRB constituted less than 6% and no increase was observed during nitrate addition (indicating that other, unidentified, NRB populations were present). After 1–3 months without significant H2S production (3.5–5.5 months with nitrate), the SRB population collapsed, the fraction being reduced to 9–25%. The dominant SRB strain in the column, which constituted on average 94% of the monitored SRB population, was partly/completely inhibited by 50/75 µM nitrite in batch culture tests. Similar nitrite concentrations (50–150 µM) were detected in the column when the H2S level declined, indicating that nitrite inhibition was the main cause of H2S elimination. The results from this study indicate that nitrate/nitrite can be used to prevent detrimental SRB activity in oil reservoirs.
Natural attenuation potential of cyanide via microbial activity in mine tailings by K. Oudjehani; G. Zagury; L. Deschênes (pp. 409-415).
Biological removal by indigenous microflora of cyanide, contained in old (6–9 years) and fresh tailings (3 months), was studied in order to assess its natural attenuation potential via biodegradation. To investigate the presence of indigenous microflora in tailings, total heterotrophic and cyanide resistant bacteria were counted using the spread-plate method. The free cyanide mineralization potential was estimated using K14CN in the presence of various unlabeled cyanide concentrations (0, 5, and 10 mg CN–/kg). The biodegradation of cyanide contained initially in the samples was also investigated by monitoring formate, formamide, ammonia and total cyanide (CNT) concentrations over 111 days. The enumeration of total heterotrophic and cyanide-resistant bacteria in old tailings showed an average population of 105 cfu/g. However, no growth was detected in fresh tailings. Nevertheless, cyanide mineralization tests indicated the presence, in both old and fresh tailings, of a cyanide-degrading microflora. In old tailings, maximum mineralization percentages of free cyanide ranging from 85% to 100% were obtained after 65 days at all concentrations tested. A mineralization percentage of 83% after 170 days was also observed in fresh tailings. No decrease of total cyanide concentration in old tailings was observed when the biodegradation of endogenous cyanide was tested whereas a significant decrease was recorded in fresh tailings after 96 days. The presence of strong metal–cyanide complexes resistant to biodegradation could explain the absence of biodegradation in old tailings. This study demonstrated the presence of an indigenous free cyanide-degrading microflora in both old and fresh tailings, and suggests that natural attenuation of cyanide in gold mine tailings is likely to occur via microbial activity.
Bioremediation of toxic chromium from electroplating effluent by chromate-reducing Pseudomonas aeruginosa A2Chr in two bioreactors by A. Ganguli; A. Tripathi (pp. 416-420).
The chromate-reducing ability of Pseudomonas aeruginosa A2Chr was compared in batch culture, with cells entrapped in a dialysis sac, and with cells immobilized in an agarose-alginate film in conjunction with a rotating biological contactor. In all three systems, the maximum Cr(VI) reduction occurred at 10 mg Cr(VI)/l. Whereas at 50 mg Cr(VI)/l concentration, only 16% of the total Cr(VI) was reduced, five spikings with 10 mg chromate/l at 2-h intervals led to 96% reduction of the total input of 50 mg Cr(VI) /l. Thus maximum Cr(VI) reduction was achieved by avoiding Cr(VI) toxicity to the cells by respiking with lower Cr(VI) concentrations. At 10 mg Cr(VI)/l, the pattern of chromate reduction in dialysis-entrapped cells was almost similar to that of batch culture and 86% of the bacterially reduced chromium was retained inside the dialysis sac. In electroplating effluent containing 100 mg Cr(VI)/l, however, the amount of Cr(VI) reduced by the cells immobilized in agarose-alginate biofilm was twice and thrice the amount reduced by batch culture and cells entrapped in a dialysis sac, respectively.